Early B cell Factor (EBF) is an essential regulator of genes in B lymphocytes. EBF drives the development of early progenitors to become B cells by regulating genes encoding the pre-B and mature B cell receptors (pre-BCR and BCR). B cell development is arrested and immunoglobulins (Ig) are not produced in mice lacking EBF. In the absence of EBF, target genes including mb-1 (Ig-1) fail to be activated and reside in hypermethylated, inaccessible chromatin. Recently, the Hagman laboratory demonstrated that EBF is required for and initiates CpG demethylation and the remodeling of nucleosomes at the mb-1 promoter during B lymphopoiesis. These epigenetic modifications are required to facilitate activation of the mb-1 promoter by other transcription factors, including Pax5. Thus, EBF is a key component of a new "functional hierarchy" of transcription factors that acts in addition to the "genetic hierarchy" identified previously. In spite of the functional importance of EBF, very little is understood concerning its three-dimensional structure. EBF and its paralogues in flies, worms and vertebrates possess highly conserved DNA-binding and helix-loop-helix (HLH) domains. Notably, the primary sequence of the EBF DNA binding domain suggests a novel structure with little relation to other types of DNA binding domains. The uniqueness of EBF is also suggested by its other highly conserved domains, which mediate its dimerization and stability. Therefore, we hypothesize that a better understanding of how EBF functions in B lymphocytes and other target tissues (adipocytes and cortical neurons), will come from knowledge of its three-dimensional structure. In the two Aims of this proposal, X-ray crystallography and other methods will be used to obtain new structural information about EBF and EBF-DNA complexes. This information will be validated in vivo and will guide the analysis of mutated EBF proteins in biological assays that assess EBF-dependent chromatin remodeling, protein:protein interactions and transcriptional activation. Together, these studies will greatly facilitate our understanding of EBF and its regulation of genes in B lymphocytes. These studies will be broadly applicable to understanding how pioneer transcription factors initiate chromatin remodeling in lymphoid as well as other cell types. Public Health Relevance: The work is relevant to the public health, because it will lead to a better understanding of how genes are regulated and may be manipulated as part of future therapies. Specifically, many diseases are the result of incorrect gene expression, and the ability to reverse the epigenetic codes that cause gene inactivity, such as DNA methylation, will provide promising approaches for therapeutic intervention. However, to achieve such therapies, it will be necessary to understand at a molecular level how a transcription factor such as EBF initiates transcriptional activity of lymphoid genes through inducing DNA demethylation of the mb-1 promoter.